PROJECT SUMMARY Differentiated vascular smooth muscle cells (VSMCs) are genetically programmed to proliferate and migrate at low rate while carrying out contractility for adult vessel homeostasis. VSMCs are not terminally differentiated and can undergo phenotypic switching to a dedifferentiated synthetic mode in response to various pathophysiological stimuli, contributing to diverse vascular diseases such as atherosclerosis and restenosis. Due to cell surface accessibility, cell membrane regulators required for VSMC differentiation may hold attractive therapeutic potential. However, there is a paucity of information on key transmembrane regulators governing VSMC differentiation. TSPANs possess a unique signaling platform, termed as tetraspanin-enriched microdomains (TEMs) organized in cis with TSPANs and other transmembrane partners including receptor tyrosine kinases and integrin(s). TSPANs serve as membrane ?docking? molecules, exerting critical roles in diverse signal cascades. Recent genome wide association studies have specifically linked TSPAN2 to atherosclerosis and blood pressure control. Inspired by these findings, we interrogated data from multiple RNA screenings and consistently found that TSPAN2 is the sole TSPANs member significantly decreased during VSMC phenotypic modulation and in diseased vessels. TSPAN2 is enriched in VSMCs and exhibits serum response factor (SRF) /Myocardin (MYOCD)-dependent expression in vitro. New functional data shows that depletion of TSPAN2 in VSMCs attenuates contractile gene expression, while promoting VSMC proliferation and migration; forced expression of TSPAN2 blocks neointima formation in a balloon injury model. TSPAN2 interacts with and promotes degradation of CD44 protein, a prominent pathological mediator in vascular disease. In addition, TSPAN2 interacts with the laminin-binding integrin ?3?1, and ?1 integrins are essential to the maintenance of the VSMC contractile phenotype. These preliminary findings support a novel hypothesis that TSPAN2 stabilizes the VSMC contractile phenotype and suppresses vascular pathology via two distinct pathways: by promoting degradation of CD44 protein which inactivates the synthetic VSMC phenotype and interaction with integrin ?3?1 which facilitates the contractile phenotype. Three specific aims are proposed to test this hypothesis. Aim 1 will determine functions of TSPAN2 in arteriovenous fistula and atherosclerosis mouse models. Aim 2 will elucidate the mechanisms through which TSPAN2 stabilizes the VSMC contractile phenotype involving inactivation of the synthetic VSMC phenotype by promoting CD44 protein degradation and activation of the contractile phenotype by interacting with integrin ?3?1. Aim 3 will elucidate the molecular basis for the impaired TSPAN2 gene expression under pathological vascular conditions via perturbation of SRF/MYOCD pathway. Successful completion of this proposal will reveal previously unknown molecular mechanisms governing VSMC phenotypic plasticity involving the actions of key transmembrane regulators, which will potentially lead to more effective therapies for vascular diseases.